Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. CRC contains a well-defined cancer stem cell population capable of both self-renewal and differentiation into daughter cells that comprise the bulk of the tumor. It has recently been shown that normal intestinal stem cells are the cell of origin in CRC, as oncogenic mutations activating the Wnt pathway (primarily through genetic inactivation of the APC tumor suppressor) result in tumor formation only when they occur in an intestinal stem cell, and not its differentiated progeny. It is therefore a priority to understand how mechanisms underlying stem cell fate determination in normal intestinal stem cells can become deregulated and contribute to the ontogeny of CRC. Adult stem cells often undergo asymmetric cell fate determination to insure that the proper ratio of stem cells to differentiated cells is maintained during homeostasis. In neuroblasts of Drosophila melanogaster this process is governed by the activity of the Musashi RNA binding protein, whose mammalian orthologs, Msi1 and Msi2, are highly expressed in mammalian intestinal stem cells and in human CRC. We propose that deregulation of Msi proteins alters cell fate determination in CRC such that stem cells expand at the expense of their differentiated progeny, thus resulting in the promotion of aggressive, undifferentiated tumors. This hypothesis will be addressed using genetically altered mice in which Msi protein levels can be modulated in the intestinal stem cells. Initially, we will determin whether Msi gain of function is sufficient to induce stem cell-driven tumorigenesis. To this end, drug inducible Msi1 and Msi2 transgenic mice have been generated, enabling efficient induction of Msi activity with precise spatio-temporal control. To determine whether Msi activity is required for the onset and progression tumorigenesis in the APCmin/+ model of stem cell-driven intestinal cancer, conditional deletion alleles of both Msi1 and Msi2 were generated. Deletion of Msi genes at the onset or during the course of tumor progression will unequivocally determine their functional role in disease ontogeny. Finally, using an in vivo CLIP-Seq approach, we have recently identified a number of Msi1 RNA binding targets associated with regulation of the Wnt pathway and have also observed that Msi genes are activated in response to Wnt signaling in vivo. We will therefore address the hypothesis that Msi is an important effector of Wnt-mediated intestinal transformation and that Msi resides in a negative feedback loop with the Wnt signaling pathway to govern the normal balance between stem cells and their differentiated progeny in the intestine. This study will provide novel insight into the fundamental mechanisms by which stem cell-driven cancers are initiated and progress. Moreover, as cancer stem cells are often resistant to conventional therapeutics such as radiation or chemotherapy and their survival can promote tumor recurrence, we predict that our studies will identify novel therapeutic targets for the treatment of CRC and other stem cell- driven tumors. ! PUBLIC HEALTH RELEVANCE: Colorectal cancer is the third leading cause of cancer related deaths in the world and is driven by a well-defined cancer stem cell population capable of both self-renewal and differentiation. We have recently identify the Msi family of RNA binding proteins as potent regulators of developmental potency in somatic stem cells and have developed mouse models to address the hypothesis that Msi deregulation alters intestinal stem cell fate determination thereby contributing to the oncogenic transformation that leads to colorectal cancer. Our findings will identify novel points of therapeutic intervention not only in colorectal cancer, but also in a wide range of stem cell-driven epithelial cancers.